High‐affinity amide‐lanthanide adsorption to gram‐positive soil bacteria

Abstract The gram‐positive soil bacterium, Arthrobacter nicotianae, uses multiple organic acid functional groups to adsorb lanthanides onto its cell surface. At relevant soil pH conditions of 4.0–6.0, many of these functional groups are de‐protonated and available for cation sorption and metal immobilization. However, among the plethora of naturally occurring site types, A. nicotianae is shown to possess high‐affinity amide and phosphate sites that disproportionately affect lanthanide adsorption to the cell wall. We quantify neodymium (Nd)‐selective site types, reporting an amide‐Nd stability constant of log10 K = 6.41 ± 0.23 that is comparable to sorption via phosphate‐based moieties. These sites are two to three orders of magnitude more selective for Nd than the adsorption of divalent metals to ubiquitous carboxyl‐based moieties. This implies the importance of lanthanide biosorption in the context of metal transport in subsurface systems despite trace concentrations of lanthanides found in the natural environment.


INTRODUCTION
Owing to their high abundance in soils and sediments and to their highly reactive surfaces, bacteria can play an important role in the fate, transport and overall mobility of metals in surface and subsurface environments (Yee & Fein, 2002).It is understood that bacteria can co-transport these adsorbed metals through preferential flow paths in porous soil structures (McCarthy & Zachara, 1989).Relevant metals that bind to bacteria include contaminants such as uranium (Haas et al., 2001;Yung & Jiao, 2014) and cadmium (Butzen & Fein, 2019;Hatano & Tsuruta, 2017;Loukidou et al., 2005) as well as trace rare earth elements (REEs) released from natural mineral deposits and geothermal brines (Emmanuel et al., 2012;Kang et al., 2019;Takahashi et al., 2010;Wood, 2002).Although REEs are usually present at low concentrations (low ppm to ppb range), these trivalent metals may still pose a strong control over the cell surface's adsorption capability due to high-affinity REE surface complexation interactions (Andrès et al., 2003;Martinez et al., 2014;Ngwenya et al., 2010).
Although carboxyl, amine and hydroxyl groups have been documented as functional groups found on bacterial surfaces (Hong & Brown, 2006), the impact of amide groups on surface-mediated bacterial biosorption is less well established.Unlike the other important functional groups, carbonyl-based amide groups are typically neutrally charged and could thus be expected to have weaker electrostatic interactions with REEs compared to negatively charged surface ligands.Maleke et al. (2019), however, demonstrated that carbonyl-based amide groups can play an important role in europium adsorption onto the thermophilic bacterium, Thermus scotoductus.Due to the formation of a resonance structure, the delocalization of the C═O carbonyl pi-bond allows for the formation of a C─O À site (Figure 1), enabling carbonyl-based amide moieties to be potential active sorption sites (Condamines & Musikas, 1992;Cui et al., 2007;Feng et al., 1996;Gholivand et al., 2018).This process is driven by the presence of a strong Lewis acid, which can pull the electron density away from the initial C O double bond.The resultant C O À site creates a particularly favourable electrostatic interaction with the trivalent rare earth metals and other charge-dense cation metals.
To model this phenomenon, surface complexation modelling (SCM) is a useful tool to describe speciation of adsorbed metals onto reactive surfaces, incorporating numerous important factors such as aqueous speciation, surface ligand-based thermodynamics and accumulation of surface charge into the computational method (Davis & Kent, 2018).However, while past studies investigating bacteria-based REE biosorption have successfully fitted adsorption edge and isotherm data to one-or two-site surface complexation models (Markai et al., 2003;Ngwenya et al., 2010), most of these studies do not consider the carbonyl-based amide sites as readily active REE-binding surface ligands.
Here, we present a study elucidating the interfacial chemical process whereby a gram-positive bacterium preferentially adsorbs a trivalent REE cation over more commonly occurring divalent heavy metals (Fein, 2000;Fein et al., 2001).We identify the gram-positive soil bacterium, Arthrobacter nicotianae, as a model microorganism that can adsorb REEs via an amide-based complexation mechanism.Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy is used to qualitatively confirm site-specific adsorption information.In addition, a novel surface complexation model for A. nicotianae is developed that extends beyond commonly accepted surface functional groups to include carbonyl-based amide groups.The introduction of a new REE adsorption mechanism in the form of carbonyl-based amide groups enables more accurate predictions of neodymium (Nd) biosorption over a relevant range of pH and metal loading conditions available in natural soil systems.

RESULTS AND DISCUSSION
Nd adsorption mechanism using ATR-FTIR At pH 4.0, clear increases in the negative absorbance difference at 1641 and 1540 cm À1 peaks were observed at the amide I and II peaks, indicating the participation of carbonyl-based amide adsorption of Nd (Figure 2).An increase in negative absorbance difference at $1400 cm À1 was also observed, indicating carboxyl-based adsorption.At lower Nd surface excess, changes in infrared (IR) absorbance were mostly present in amide I and II and phosphodiester peaks.At higher Nd surface excess, changes in the carboxyl peaks were observed with minimal changes in amide peaks (Figure 2B).A splitting effect at the 1406 cm À1 de-protonated carboxyl peak indicated both the decrease of free de-protonated carboxyl sites at 1408 cm À1 and the increase in new Nd-carboxyl bonds as identified by the ingrowing 1384 cm À1 peak.This splitting phenomenon due to the formation of new metal-surface ligand complexes has also been observed in the FTIR measurements of eggshell biosorption of divalent Pb and Zn ions (Pranata Putra et al., 2014) and zeolite coordination of Cu + cations (Zdravkova et al., 2015).Similar splitting effects were observed for phosphodiester and phosphate peaks, strongly suggesting the binding of Nd onto these functional groups.The increased absorbance intensity of the splitting patterns found in the difference spectra at higher surface excess indicated that as more Nd was added to the cell suspension, more Nd-surface ligand complexes were formed with amides and phosphatebased moieties.Similar trends were also observed for pH 6.0 (Figure S1), though the relative magnitudes were quite different.Unlike the pH 4.0 spectra, IR absorbance results from the pH 6.0 condition were dominated by a stronger absorbance change in the 1646 cm À1 amide I peak.

Modelling high-affinity sorption sites of A. nicotianae
Initial bacterial cell surface characterization of protonation states is conducted using titration experiments and SCM (Figure S2).Nd adsorption data for 0.1 M ionic strength and 25 C temperature were pooled together using pH 4.0, 5.0 and 6.0 adsorption isotherms collected from Park et al. (2020) and pH 4.0 and 6.0 Nd adsorption isotherms conducted in the present study.A detailed explanation of the experimental and modelling methods can be found in the Supporting Information.Experimentally measured surface excess and distribution coefficient values determined in the present work were consistent with Park et al. (2020) (Figure 3).Increases in pH yielded greater maximum adsorption capacity as well as higher surface selectivity for Nd as presented by pH-dependent increases in both surface excess and distribution coefficients.Modelled Nd-stability constants indicate high-affinity site types mostly in the form of phosphate-based ligands and carbonyl-based amide moieties.Both 1:1 phosphodiester and phosphoryl-Nd surface complexation reactions have high log 10 K binding constants of 6.32 ± 0.07 and 6.69 ± 0.08, respectively, and carbonyl-based amide-Nd stability was determined in this study to be log 10 K of 6.41 ± 0.23 (site density and stoichiometry available in Tables S1 and S2).In contrast, carboxyl-Nd adsorption was modelled as having a much lower log 10 K = 4.65 ± 0.75 binding constant.This low Nd stability constant indicates that carboxyl-based surface ligands on bacterial surfaces are non-selective site types with relatively weak thermodynamic driving force to electrostatically bind trivalent cations such as Nd.
The high-stability constant calculated for amide-Nd stability can be explained by the strong Lewis acid nature of trivalent Nd and the ability for a large concentration of neutrally charged C O linked amides to form C O À sites from the resonance-induced delocalization of pi-bond electrons.This mechanism is qualitatively supported by our ATR-FTIR difference spectra, whereby large absorbance changes in the carbonylbased amide I peak are observed when the bacteria is loaded with increasing amounts of Nd.This resonancebased adsorption mechanism implies the presence of a potentially high selectivity site type for lanthanide surface complexation.Because ion-amide complexes increase in strength with higher ionic potential cations (Feng et al., 1996), trivalent lanthanides pose the strongest control over the complexation of these site types in comparison with other co-occurring divalent metals, such as Zn 2+ and Cd 2+ , present in soils.

CONCLUSIONS
Future research directions for this work include quantitative ATR-FTIR and x-ray absorption spectroscopy analyses to further elucidate the multi-site competitive surface complexation reactions taking place.Challenges for this advancement include peak deconvolution methods for FTIR, particularly at carboxyl and phosphodiester peaks that split due to the formation of new Nd-surface ligand complexes.Although this work focuses on gram-positive bacteria, further research is still needed on other soil microorganisms, such as gram-negative bacteria and fungi, and studies in realworld soil matrices have yet to be conducted.This study presents a new approach in quantifying a highaffinity amide-Nd reaction mediated by a reactive soil microbial cell surface cultured in lab setting.

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I G U R E 1 Resonance structure enabling neodymium (Nd) complexation to carbonyl-based amide moieties.

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I G U R E 2 (A) Normalized ATR-FTIR spectra of wet cell suspensions of Arthrobacter nicotianae at pH 4.0 and (B) associated difference spectra at varying Nd metal loadings.Thin and thick lines indicate non-smoothed and smoothed ATR-FTIR measurements, respectively.ATR-FTIR, attenuated total reflectance Fourier Transform Infrared; Nd, neodymium.F I G U R E 3 Pooled (A) surface excess and (B) distribution coefficient data of neodymium adsorption isotherms from Park et al. (2020) (filled circles) and results from this study (open circles).